P450 genes are under complex control. Several levels of regulation exist that govern inducibility, tissue specific expression and developmentally- programmed gene activation. Rat liver has served as a model for P450 gene expression. We have been studying the CYP2E1 and CYP2C6 genes that are expressed in liver and are activated at distinct stages of development. CYP2E1 transcription is markedly activated within one day after birth, while CYP2C6 is not expressed until rats reach puberty. By a series of in vitro transcription and DNA-binding assays, CYP2E1 was found to be controlled by the hepatocyte-enriched transcription factor HNF-1 alpha (hepatocyte nuclear factor-1 alpha) that binds to a region of DNA upstream of the transcription start site of CYP2E1. This factor is expressed just prior to birth and is under control of a second factor, designated HNF-4 indicating that CYP2E1 is part of a regulatory cascade. The CYP2C6 gene was found to be under control of the hepatocyte-enriched transcription factor DBP. This factor was found to bind to the D site of the serum albumin gene and to control its transcription in adult rats. DBP expression occurs at the onset of puberty in male and female rats as does the expression of CYP2C6. By use of trans-activation transfection assays using the CYP2C6 promoter to drive the chloramphenicol acetyltransferase gene and the DBP cDNA under control of the cytomegalovirus promoter, we demonstrated that DBP could activate tran- scription of the CYP2C6 promoter. Recombinant DBP was also able to specifically bind to a segment of DNA upstream of the CYP2C6 transcription start site.